Team:Paris/August 5

From 2008.igem.org

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{{Paris/Calendar_Links|August 4|August 6}}
{{Paris/Calendar_Links|August 4|August 6}}
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== ==
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== '''Amplification of Promotors of interest (FliA, FliL, FlgA, FlgB, FlhB, FlhDC)'''==
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We performed PCR on   to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.
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''We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.''
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=== PCR Protocol ===
 
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=== '''PCR Protocol''' ===
 +
 +
* '''Preparation of the templates''' :--> Resuspend of 1 colony of MG1655 in 100µl of water.
 +
 +
* '''List of Oligos''' :
 +
{|
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|- style="background: #649CD7;"
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! colspan="5" style="background: #649CD7;" | Table of Oligos
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|- style="background: #649CD7; text-align: center;"
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|width=5%| Number
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|width=10%| Name
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|width=40%| Sequence
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|width=5%| Length
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|width=5%| Tm(°C)
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|width=35%| Comments
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|- style="background: #dddddd;" 
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| style="background: #D4E2EF;" |O100
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| FlgA-F
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| GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCATATCTCCTCCGCAGGTATCAAAAT
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| 58
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|
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|
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O101
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| FlgA-R
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| GTTTCTTCCTGCAGCGGCCGCTACTAGTAACAGTATCGCGATGATCGCCACGCTACGT
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| 58
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|
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|
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O102
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| FlgB-F
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| GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACAGTATCGCGATGATCGCCACGCTACG
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| 58
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|
 +
|
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O103
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| FlgB-R
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| GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGCATATCTCCTCCGCAGGTATCAAAATT
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| 58
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|
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|
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O108
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| FlhB-F
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| GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCACGTCATATCAGGCGGTCTGATAAGG
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| 58
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|
 +
|
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O109
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| FlhB-R
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| GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTTTGTCGTCGCTCTCGTCAGACACGTC
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| 58
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|
 +
|
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O111
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| FlhDC-Total-F
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| GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTCATTTTTGCTTGCTAGCGTACGGAAAA
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| 58
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|
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|Amplify the both OmpR binding site
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O113
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| FlhDC(nu)-R
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| GTTTCTTCCTGCAGCGGCCGCTACTAGTACAGAATAACCAACTTTATTTTTATG
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| 54
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|
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|Don't amplify the natural rbs of FlhD (only promoter)
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O124
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| FliL-F
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| GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCAGCGAGAGGCTGTTGGTATTAATGACT
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| 58
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|
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|
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|- style="background: #dddddd;"
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| style="background: #D4E2EF;" | O125
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| FliL-R
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| GTTTCTTCCTGCAGCGGCCGCTACTAGTACCAGCGATGAAATACTTGCCATGCGATTT
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| 58
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|
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|
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|}
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 +
 +
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* '''Preparation of PCR mix''' :
''For each samples,''
''For each samples,''
* 1 µl dNTP
* 1 µl dNTP
* 10 µl Buffer Phusion 5x
* 10 µl Buffer Phusion 5x
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* 2,5 µl VR2 (O18)
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* 2,5 µl Oligo_F
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* 2,5 µl VF (O19)
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* 2,5 µl Oligo_R
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* 1µl template
* 1 µl Phusion
* 1 µl Phusion
* 50 µl qsp H2O (33µl)
* 50 µl qsp H2O (33µl)
 +
 +
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* Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Revision as of 13:07, 6 August 2008

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Contents

Amplification of Promotors of interest (FliA, FliL, FlgA, FlgB, FlhB, FlhDC)

We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.


PCR Protocol

  • Preparation of the templates :--> Resuspend of 1 colony of MG1655 in 100µl of water.
  • List of Oligos :
Table of Oligos
Number Name Sequence Length Tm(°C) Comments
O100 FlgA-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCATATCTCCTCCGCAGGTATCAAAAT 58
O101 FlgA-R GTTTCTTCCTGCAGCGGCCGCTACTAGTAACAGTATCGCGATGATCGCCACGCTACGT 58
O102 FlgB-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACAGTATCGCGATGATCGCCACGCTACG 58
O103 FlgB-R GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGCATATCTCCTCCGCAGGTATCAAAATT 58
O108 FlhB-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCACGTCATATCAGGCGGTCTGATAAGG 58
O109 FlhB-R GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTTTGTCGTCGCTCTCGTCAGACACGTC 58
O111 FlhDC-Total-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTCATTTTTGCTTGCTAGCGTACGGAAAA 58 Amplify the both OmpR binding site
O113 FlhDC(nu)-R GTTTCTTCCTGCAGCGGCCGCTACTAGTACAGAATAACCAACTTTATTTTTATG 54 Don't amplify the natural rbs of FlhD (only promoter)
O124 FliL-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCAGCGAGAGGCTGTTGGTATTAATGACT 58
O125 FliL-R GTTTCTTCCTGCAGCGGCCGCTACTAGTACCAGCGATGAAATACTTGCCATGCGATTT 58


  • Preparation of PCR mix :

For each samples,

  • 1 µl dNTP
  • 10 µl Buffer Phusion 5x
  • 2,5 µl Oligo_F
  • 2,5 µl Oligo_R
  • 1µl template
  • 1 µl Phusion
  • 50 µl qsp H2O (33µl)


  • Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29


Electrophoresis

When the PCR cycles were finished,

  • 10 µL of 6X loading dye were added
  • The samples were then loaded (2 x 30 µL per sample) on a 1,5% agarose gel.


After electrophoresis, the bands corresponding to MP 100 and MP 120 were excised and purified using the QIAquick DNA Gel Extraction Kit (QIAGEN). The elution was made in 50 µL of water. Because the intensity of the band corresponding to MP 120 was very low, we only continued with MP 100. MP 100 was digested by EcoRI & SpeI (Forward Insert) or by XbaI & PstI (D100 : Backward Insert).

KR000102.jpg
KR000104.jpg
KR000106.jpg

DNA Digestion

Digestion reaction (total volume : 50 µL)

  • 25 µL DNA
  • 5 µL buffer 2 (10X)
  • 2 µL enzyme 1
  • 2 µL enzyme 2
  • 0.5 µL BSA (100X)
  • 15,5 µL water
  • Incubate 2 hours at 37°C
  • The samples (2 x 30 µL per sample) were then analysed by electrophoresis.


Electrophoresis (bis)

The sequence of MP 100 (B0034) digested by EcoRI & SpeI (35 bp) or XbaI & PstI (34 bp) was too short and we didn't manage to visualise it on the gel.

Conclusion : small parts like B0034 can't be cloned as an insert.

Retrieved from "http://2008.igem.org/Team:Paris/August_5"